2008 Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Pro

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Infected with SARS-CoV
Protein Causes Severe Pneumonia in Mice
Coronavirus (SARS-CoV) Nucleocapsid
Respiratory Syndrome (SARS)-Associated
Prior Immunization with Severe Acute

Matsushima and Michinori Kohara
Hisatoshi Shida, Minoru Kidokoro, Kyosuke Mizuno, Kouji
Hidenori Suzuki, Katsuo Karamatsu, Yasuhiro Yasutomi,
Satoshi Sekiguchi, Kouichi Morita, Tsunekazu Hishima,
Inoue, Misako Yoneda, Shoji Yokochi, Ryoichi Kase,
Fumihiko Yasui, Chieko Kai, Masahiro Kitabatake, Shingo

http://www.jimmunol.org/content/181/9/6337
doi: 10.4049/jimmunol.181.9.6337
2008; 181:6337-6348; ;
J Immunol

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Prior Immunization with Severe Acute Respiratory Syndrome
(SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein
Causes Severe Pneumonia in Mice Infected with SARS-CoV1

Fumihiko Yasui,* Chieko Kai,‡ Masahiro Kitabatake,2* Shingo Inoue,� Misako Yoneda,‡

Shoji Yokochi,*§ Ryoichi Kase,* Satoshi Sekiguchi,* Kouichi Morita,‡ Tsunekazu Hishima,¶

Hidenori Suzuki,† Katsuo Karamatsu,# Yasuhiro Yasutomi,# Hisatoshi Shida,**
Minoru Kidokoro,†† Kyosuke Mizuno,‡‡ Kouji Matsushima,§ and Michinori Kohara3*

The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneu-
monia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized
intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simul-
taneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES)
7–8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the
control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as
LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that ex-
pressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e.,
LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N � LC16mOrVV-S, LC16mOrVV-M �
LC16mOrVV-S, or LC16mOrVV-E � LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-
immunized mice and LC16mOrVV-N � LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-
N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-�, IL-2) and Th2 (IL-4, IL-5) cytokines and
down-regulation of anti-inflammatory cytokines (IL-10, TGF-�), resulting in robust infiltration of neutrophils, eosinophils, and lym-
phocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response
against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase
our understanding of the pathogenesis of SARS.
The Journal of Immunology, 2008, 181: 6337–6348.
F

rom November 2002 to July 2003, an outbreak of severe
acute respiratory syndrome (SARS),4 which originated in
China, spread worldwide, resulting in 8098 cases with 774
deaths
(http://www.who.int/csr/sars/country/en/index.html).
Pa-

tients with SARS usually develop high fever followed by severe
clinical symptoms, which include acute respiratory distress syn-
drome with diffuse alveolar damage, and ultimately death. A novel
type of coronavirus (CoV), termed SARS-associated CoV (SARS-
CoV), was identified as the etiologic agent of SARS (1–3). The
genome of SARS-CoV is a single strand of positive-sense RNA of
�30 kb in length with 14 putative open reading frames, which
encode nonstructural replicase polyproteins and several structural
proteins, including spike (S), envelope (E), membrane (M), and
nucleocapsid (N) proteins (4). The S protein of SARS-CoV, like
the S proteins of other CoVs, plays an important role in the first
step of viral infection by binding to a host cell receptor. Angio-
tensin-converting enzyme 2 was identified as the host receptor for
SARS-CoV (5). Angiotensin-converting enzyme 2 is abundantly
expressed in the epithelia of the lung and small intestine and may
mediate SARS-CoV entry in humans (6). Although intensive in-
vestigations rapidly unraveled the sequence of the SARS-CoV ge-
nome and its receptor in humans, the precise molecular mechanism
underlying the development of SARS is not fully understood.
The possible roles of host anti-SARS-CoV immune responses
have been suggested in severe clinical cases. The uncontrolled
release of immune mediators, called a “cytokine storm,” has been

*Department of Microbiology and Cell Biology, †Laboratory of Electron Microscopy,
The Tokyo Metropolitan Institute of Medical Science, ‡Laboratory Animal Research Cen-
ter, The Institute of Medical Science, §Department of Molecular Preventive Medicine,
School of Medicine, The University of Tokyo, and ¶Department of Pathology, Tokyo
Metropolitan Komagome Hospital, Tokyo, Japan; �Department of Virology, Institute of
Tropical Medicine, Nagasaki University, Nagasaki, Japan; #Laboratory of Immunoregu-
lation and Vaccine Research, Tsukuba Primate Research Center, National Institute of
Biomedical Innovation, Ibaraki, Japan; **Division of Molecular Virology, Institute for
Genetic Medicine, Hokkaido University, Sapporo, Japan; ††Third Department of Virol-
ogy, National Institute of Infectious Diseases, Musashimurayama, Japan; and ‡‡The
Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan

Received for publication January 23, 2008. Accepted for publication August 23, 2008.

The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by a Grant for Research on Emerging and Re-emerging
Infectious Diseases from the Ministry of Health, Labor and Welfare, Japan, by the 21st Cen-
tury Centers of Excellence program on Global Strategies for Control of Tropical and Emerging
Infectious Diseases at Nagasaki University, and by the Ministry of Education, Culture, Sports,
Science and Technology of Japan. Strategic cooperation to control emerging and re-emerging
infections is funded by the Special Co-ordination Fund for Promoting Science and Technology
of the Ministry of Education, Culture, Sports, Science and Technology.

2 Current address: Department of Immunology, Graduate School of Medicine, Kum-
amoto University, 1-1-1 Honjo, Kumamoto, 860-8556, Japan.

3 Address correspondence and reprint requests to Dr. Michinori Kohara, Department
of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical
Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. E-mail ad-
dress: